Due to the impermeability and short half-life, most of protein therapeutics require frequent injection. To reduce injection frequency, development of sustained release dosage forms has been a long-standing research focus since 1970s (1). In spite of extensive research efforts (2), up to now, sustained release formulation technology has succeeded in only one protein drug, recombinant human growth hormone (rhGH). The major roadblocks are invariably the protein instability in formulation process and at the site of release (3, 4) as well initial burst and incomplete release. Various strategies to improve protein stability in microencapasulation have been reported in last decades (3, 5, 6). Many of these approaches, however, only address one or some issues, leaving others unsolved or even creating new problems. Some methods are feasible for only specific proteins, and some reports are contradictory to each other due to different focal points of researchers. For example, for the only commercially available long-acting protein, sustained release rhGH, the protein was stabilized by forming complex with zinc ions (7) based on that natural hGH forms complex with zinc in secretory granules (8). When zinc was co-encapsulated with another protein, erythropoietin (EPO) for example, up to 40% of released proteins was aggregated (9), which could result in unwanted immunogenisity. In order to protect proteins from organic solvents used in microencapsulation, sugars, inorganic salts or other conceivable excipients are used to preformulate proteins into solid particles prior to microencapsulating them into degradable polymer microspheres through a solid-in-oil-water (S-O-W) emulsification process (7, 9, 10). These excipients often resulted in considerable burst release due to strong osmotic pressure created by their high solubility (11) and rapid dissolution (12). When highly soluble ammonium sulfate was used to stabilize EPO in microencapsulation, burst release accounted up to 55% of total drug (9).
Cleland and Jones studied the effects of various excipients on protection of rhGH and interferon γ (IFN-γ) in water-in-oil-in-water (W-O-W) and S-O-W encapsulation processes, and found that mannitol or trehalose were the best in preventing proteins from aggregation during microencapsulation process were prevented (6). Sanchez et al. examined the protection effects of similar excipients for another protein, tetanus toxoid, and found dextran, that was ineffective for recovering rhGH and IFN-γ in Cleland and Jones report, showed best protection for the protein (based on ELISA) at the release phase under a hydrated condition (10). It seems that small sugars offer better protection in dehydration steps (drying), while polysacchrides are more effective in a hydrated step (release) (13). A burst release of 60% of total loading was observed from dextran included PLGA microspheres prepared by Sanchez et al. This burst release may be attributed to the particle size of the co-lyophilized protein-excipient particles (14, 15).
The size of pre-formed protein particles plays an important role in a S-O-W process. Morita et al. demonstrated that when the mean diameter of solid protein particles increased from 5 to 20 μm, the initial release almost doubled, and encapsulation efficiency dropped from 80% to 20% (15). Cleland et al. discussed different approaches for reducing protein particle size for a S-O-W process (6). Homogenizing a lyophilized protein-excipients powder in organic solvents can only result particles above 10 □m in diameter, while milling the powders to smaller size may cause protein denature due to the shears and heat generated (6). Spray drying may produce protein particles to desired size, but shear and heat at atomization as well as the presence of air-liquid interface may cause denaturation (6, 16). Moreover, surfactants must be used in spray drying and spray freeze-drying that facilitate contact and interaction between proteins and dichloromethane (the solvent most frequently used in microencapsulation) (6). Maa et al. reported that complexation of rhGH with zinc prior to spray drying can effectively prevent aggregation of the protein (16). Again, zinc complexation can denatrue proteins other than rhGH (9). Morita et al. prepared fine protein particles by a freezing-induced precipitation with a co-solution of proteins and PEG (15, 17). But the protein particles still have to be exposed to organic solvents directly during microencapsulation. Direct contact of unprotected proteins with PLGA will cause incomplete release by strong adsorption of the proteins on the internal surface of the polymer matrix (18). To avoid the hydrophilic-hydrophobic interface, aqueous two-phase systems were used for preparing polysaccharide particles (19, 20). However, the dispersed phases need to be stabilized by covalent or ionic cross-linking, another potential cause for protein denaturation.
For sustained release of delicate proteins, an approach that can address all these important issues is highly desired. Due to the long-standing difficulties discussed above, it is unlikely that this task can be accomplished with the existing approaches. Microencapsulation strategies based on new scientific concepts are required.
In one of our previous patent application, we have reported (as the first time according to best of our knowledge) a unique microencapsulation system, stable polymer aqueous-aqueous emulsion (24). This system differs from conventional emulsions in that both the dispersed and continuous phases are aqueous. The system is also different from so-called polymer aqueous two-phase systems that form two block phases in absence of continuous mixing. This emulsion is stable for up a week without any (covalent or ionic) cross-linking treatment. Due to these unique characteristics, delicate therapeutics such as proteins, liposomes or live viruses can be loaded into the droplets of this emulsion under a condition free of chemical or physical hazards such as organic solvents, concentrated salts, extreme pH, crosslink agents, high shear stress, high interfacial tension and high temperature. By freeze-drying or other drying methods, dispersed phase of the emulsion can form glassy particles of defined shape and uniform size for various delivery purposes (inhalation or sustained release). Our previous work has established the proof-of-principle that all the stability problems raised in protein microencapsulation, such as the processes of protein loading, drying, storage and release (3), can be addressed using this unique system. In addition, all the ingredients used are those proven for injection into human.
This present application further demonstrates applications of this stable aqueous-aqueous emulsion system in delivery of protein drugs. Proteins can be loaded into the dispersed phase of the aqueous-aqueous emulsion system and form glassy particles by freeze-drying thereafter. The entire preparation process is free of any chemical physical hazards. Protein activity can be well preserved during this preparation process. Proteins loaded in the glassy particles made via the emulsion system (called AqueSphere(s) hereafter) showed strong resistance to organic solvents, prolonged activity in hydrated state at 37° C., as well as linear release profile with minimal burst and incomplete release when being further loaded in degradable polymer microsphere.